The present invention relates to a process for permanently coating the inner surface of columns, capillaries and microchannel systems with hydroxylic polymers. Such interior coating is effected by treating the surface to be coated with a solution of a cross-linking reagent and a solution of the polymer, which results in an immobilization of the polymer on the capillary surface. The process described is particularly suitable for the coating of fused silica (FS) capillaries and systems having microfluidic structures for capillary electrophoresis with a highly hydrophilic polymer, such as polyvinyl alcohol. The invention further relates to the thus prepared columns and capillaries, and their use in capillary electrophoresis and related techniques.
Methods of capillary electrophoresis (CE) can be used to obtain separations of mixtures of various compounds by differential migration of the analytes in an electric field. The separations are usually performed in capillaries of fused silica filled with a, mostly aqueous, electrolyte. The inner surfaces of such capillaries have the following properties in their application in capillary electrophoresis:
Due to the acidic silanol groups of the fused silica material, there is an electroosmotic flow (EOF) towards the cathode which depends on the pH value of the electrolyte. However, in uncoated fused silica capillaries, the absolute value of the EOF can highly vary, which significantly deteriorates the precision and reproducibility of analyses in capillary electrophoresis.
The fused silica surfaces exhibit a highly adsorptive behavior towards many compounds. In particular, basic compounds and large biopolymers, such as proteins, are strongly adsorbed from aqueous solutions. This deteriorates the quality and resolution of a capillary-electrophoretic separation of such compounds considerably or even makes it impossible.
The properties of such capillaries can be changed in a well-aimed manner by providing the capillary surface with a suitable coating. Coated fused silica capillaries are employed in capillary electrophoresis for the following reasons:
For a manipulation of the electroosmotic flow: By coating the surface, both the absolute value and the direction of the EOF can be changed. A CE separation can be optimized thereby with respect to resolution and analysis time. The constancy of the EOF is mostly higher in coated capillaries as compared to untreated fused silica capillaries, which results in improved reproducibility of CE analyses.
To suppress the interactions between analytes and the wall, coatings are employed which exhibit a low adsorptivity towards the compounds to be examined. Higher separation efficiencies of adsorptive compounds, such as proteins, and thus a better resolution is thereby achieved in capillary-electrophoretic separations.
A suitable inner coating of capillaries for CE should have the following properties: i) a constant EOF over a broad pH range; ii) long-term stability towards a wide variety of electrolytes; iii) the adsorptivity of the coating should be as low as possible towards a wide variety of analytes.
Fused silica capillaries can be very easily coated with cationic polymers, such as Polybrene(copyright) or polyvinylamine [M. Chiari, L. Ceriotti, G. Crini, and M. Morcellet; J. Chromatogr. A 836(1999) 81], because these are strongly adsorbed to the contrarily charged fused silica surfaces. Due to the reversed, anodic EOF, these capillaries are particularly suitable for the accelerated analysis of highly mobile anions.
The use of hydrophilic non-ionic capillary interior coatings, such as polyacrylamide or polyethylene glycol coatings, has proven particularly useful in capillary electrophoresis, because such coatings both significantly reduce the EOF and suppress the adsorption of basic compounds and especially proteins to the capillary wall. Different methods have been developed for chemically binding hydrophilic molecules to a fused silica capillary surface, mostly after previous silanization (S. Hjerten; J. Chromatogr. 347 (1985) 191; G. M. Bruin, J. P. Chang, R. H. Kuhlmann, K. Zegers, J. C. Kraak and H. Poppe; J. Chromatogr. 471 (1989) 429; K. A. Cobb, V. Dolnik and M. Novotny; Anal. Chem. 62 (1990) 2478; A. Malik, Z. Zhao and M. L. Lee; J. Microcol. September 5 (1993) 119).
Among all the capillary coatings described in the literature, those based on polyvinyl alcohol (PVA), which has to be considered the most hydrophilic polymer, have proven to exhibit a particularly good performance. In capillaries coated with PVA, extraordinarily high separation efficiencies could be achieved, especially for proteins. In addition, the electroosmotic flow is suppressed over a broad pH range and very stable (M. Gilges, H. Kleemixcex2 and G. Schomburg; Anal. Chem. 66 (1994) 2038).
The preparation of capillaries coated with polyvinyl alcohol is relatively difficult to perform with conventional methods. This is due to the particular properties of polyvinyl alcohol: i) Polyvinyl alcohol is soluble only in water, and therefore classical chemical reactions in organic solvents for fixing the polymer to the capillary surface, such as by silanization, are not possible. ii) Due to their high hydrophilicity, aqueous solutions of polyvinyl alcohol will wet the fused silica capillaries but poorly; therefore, it is difficult to produce homogeneous films of polyvinyl alcohol on the capillary surfaces by adsorption alone. To date, the following methods have been described for producing PVA-coated capillaries:
1) Gilges and Schomburg (M. Gilges, H. Kleemixcex2 and G. Schomburg; Anal. Chem. 66 (1994) 2038; DE 42 30 403 A1) have applied a PVA film to a capillary wall by adsorption of polyvinyl alcohol from an aqueous solution to the fused silica surface. Thus, a capillary filled with the PVA solution was emptied very slowly in a nitrogen flow using overpressure. Only by carefully and very slowly emptying the capillary, a homogeneous polymer film could be produced on the surface despite the poor wetting of the surface by the polymer solution. In the original publication (Anal. Chem. 66 (1994) 2038), a flow-restricting capillary was coupled to the capillary to be coated for a controlled emptying.
The immobilization of the polymer film, which is water-soluble at first, was then effected by heating in a gas flow. Polyvinyl alcohol is thereby converted to a pseudocrystalline state and thus insoluble in water.
With this method, stable polyvinyl alcohol coatings could be applied to the capillaries without expensive multistep chemical reactions. However, since the polymer film is applied in this method from a polymer solution which wets the fused silica wall but very poorly, this part of the process is rather error-prone.
2) Shieh (U.S. Pat. No. 5,605,613) was able to prepare PVA-coated capillaries in a multistep process. Thus, a hydrophobic intermediate layer was first bound to the fused silica surface through Sixe2x80x94Oxe2x80x94Si linkages. Polybutadienyltriethoxysilane was mentioned as a preferred reagent. Thereafter, the monomer vinyl acetate is bound to this layer through its vinyl functionality by free-radical polymerization. Subsequently, the polyvinyl acetate covalently bonded to the intermediate layer is hydrolyzed and thus converted to polyvinyl alcohol.
The procedure described by Shieh is based on a lengthy multistep reaction. The first step is based on a silanol derivatization and is therefore highly dependent on the concentration of silanol groups on the fused silica surface and thus on the respective capillary preparation process and/or the chosen preliminary treatment of the capillary.
3) The method described by Karger (U.S. Pat. No. 5,840,388) is very similar to that described by Shieh. An intermediate layer which will react with the monomer vinyl acetate is also first applied through Sixe2x80x94Oxe2x80x94Si linkages by a silanization reaction in organic solvents, preferably using vinyltrimethoxysilane. Then, as in Shieh, covalently bonded polyvinyl acetate is generated in a free-radical copolymerization reaction of the intermediate layer with vinyl acetate. Then, following hydrolysis, polyvinyl alcohol covalently bonded to the silica surface is obtained by analogy with Shieh.
It has been the object of the invention to provide a quick and reliable process for coating capillaries with hydroxylic polymers, preferably polyvinyl alcohol, and their use in electromigrative separation methods.
This object of permanently coating a capillary with a polymer, preferably polyvinyl alcohol, is achieved by chemically cross-linking the dissolved polymer during the coating process and thus immobilizing it on the capillary wall. Thus, a dissolved bi- or multifunctional reagent, preferably a dialdehyde, such as glutaraldehyde, is transferred into the capillary. Thereafter, a plug of the polymer solution, preferably a polyhydroxy compound, such as polyvinyl alcohol, is forced through the capillary, for example, using a pressurized gas, such as nitrogen or other gases usual in a laboratory, e.g., helium, argon, hydrogen, or by means of a mercury plug. The reaction occurring between the polymer solution and the cross-linking reagent enables a good wetting of the capillary wall with the polymer solution and at the same time immobilizes the produced polymer layer on the capillary wall. The thus generated very stable polymer coatings can be employed immediately after drying by additional flushing with gas, and exhibit the following properties in capillary electrophoresis: suppressed EOF both in acidic and alkaline pH ranges, very high separation efficiencies for adsorptive analytes, such as basic proteins, long-term stability upon use of aqueous and non-aqueous electrolytes, stability towards highly acidic and alkaline rinsing solutions, such as diluted HCl or NaOH solutions.
This process enables PVA-coated capillaries of high quality to be prepared very quickly and with a high reliability. The coating process takes a few minutes, and the drying of the capillaries is effected in a nitrogen flow over several hours. The drying time can be significantly reduced by increasing the temperature so that the capillaries can be available in less than an hour.
Capillaries coated with PVA and glutaraldehyde as a cross-linking reagent in accordance with the present process can be prepared with substantially higher reliability as compared to the analogous process with no cross-linking reagent (with exclusively thermal immobilization). FIG. 1 shows the separation efficiencies achievable in CE separations of basic proteins for different capillaries prepared successively. If glutaraldehyde is used as the cross-linking reagent, very high separation efficiencies (N greater than 400,000) for the strongly basic protein cytochrome c can be achieved in over 80% of the capillaries prepared.
However, if the coating is effected without the cross-linking reagent, separation efficiencies N of greater than 400,000 are obtained in only 20% of the capillaries prepared. In 30% of the capillaries thus prepared (Nos. 1, 5, 12, 15), no signal can be obtained for the strongly adsorptive protein, because it was irreversibly adsorbed as with uncoated capillaries.
In addition to classical capillaries, surfaces of systems having microfluidic structures, such as microchannels of a capillary electrophoresis chip (CE chip), can also be coated with the present process. Thus, it becomes possible to suppress the electroosmotic flow in CE chips and to prevent the adsorption of analytes such as proteins. By selectively coating individual microchannels of a more complex system while others remain uncoated, electroosmotically induced liquid flows in networks of microchannels can be manipulated in a well-aimed way. Thus, for CE chips with crossed microchannels, it is possible to coat only the main channel relevant to the separation and thus to enable the analysis of adsorptive proteins while the injection channel remains uncoated and enables electroosmotic injection in the direction towards the cathode.
The present process for the preparation of polymer-coated capillaries is characterized by the following properties:
Since the process is based on chemical cross-linking of the polymer and chemical binding of the polymer to the capillary wall is not required, it can be applied to a wide variety of capillary materials, such as glass, fused silica, various plastics, such as Teflon, pEEK, polyacrylates, and it is also applicable to channels having different structures, such as classical round capillaries, (rect)angular channels/capillaries and microchannels in microfluidic systems (such as CE chips).
The process is easy to perform.
The process is very quick.
The capillaries obtained can be repeatedly prepared in a very high quality (few rejections).
The coated capillaries prepared by this process have the following properties:
They can be repeatedly employed for separations.
They allow for CE separations of highly adsortive compounds such as proteins in acidic, neutral and alkaline electrolytes.
The electroosmotic flow is highly reduced over a broad pH range, at least from pH 2 to pH 10, and is highly constant.
The chemically immobilized PVA coatings have proven stable under the conditions of CE.
The capillaries are especially suitable for the following applications: CE separations of basic and acidic compounds, capillary gel electrophoresis, isoelectric focusing, CE separations with very low ionic strengths of the electrolyte, and CE separations with organic solvents as electrolytes, such as for the coupling of capillary electrophoresis with mass spectrometry.
The capillaries and microfluidic systems prepared according to the invention can be employed, inter alia, for the dosage of samples to be analyzed, e.g., in mass spectrometry, and for microsynthetic methods in chemistry.